Title

The interaction of alpha-conotoxins PnIA and MII with alpha3beta2 neuronal nicotinic acetylcholine receptors

Date of Award

2005

Availability

Article

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Molecular and Cellular Pharmacology

First Committee Member

Charles W. Luetje, Committee Chair

Abstract

alpha3beta2 neuronal nicotinic receptors are at least 1000-fold more sensitive to blockade by alpha-conotoxin-PnIA than are alpha2beta2 receptors. A series of chimeric subunits, formed from portions of alpha2 and alpha3, were coexpressed with beta2 in Xenopus oocytes and tested for toxin sensitivity. Determinants of toxin sensitivity were widely distributed in the extracellular domain of alpha3. Analysis of mutant receptors allowed identification of three determinants of alpha-conotoxin-PnIA sensitivity: proline 182, isoleucine 188 and glutamine 198. A homology model of the alpha3beta2 receptor extracellular domain was then generated and each residue was shown to be located on the C-loop of the alpha3 subunit, with isoleucine 188 nearest the acetylcholine-binding pocket.alpha-Conotoxin-MII was examined to find residues on the toxin that are important for conferring specificity for alpha3beta2 receptors. Each of the twelve non-cysteine residues from MII were mutated to alanine and tested for their ability to block alpha3beta2 receptors. Dose inhibition data for each mutant was acquired and IC50s were determined. Circular dichroism and protein modeling were used to confirm the structural integrity of the mutant toxins.Three residues were identified as major determinants of MII potency. Replacement of N5, P6, or H12 with alanine resulted in >2700-fold, 700-fold and ∼2700-fold losses in potency, respectively. The side chains of these residues are exposed on the surface of the toxin, suggesting that they could interact with the alpha3beta2 receptor. A decrease in pH improved MII potency, while an increase in pH decreased MII potency, suggesting that in the active form of MII, H12 is charged.Finally, a computational docking approach was used to examine the interaction between alpha-conotoxin-MII and alpha3beta2. Docking simulations place the MII toxin in the large cavity located below the C loop of the alpha3 subunit. A series of point mutations on the alpha3 and beta2 subunits were tested to confirm docking results. When aspartate 199 on the alpha3 subunit was mutated to an alanine, a tenfold loss in toxin sensitivity was observed. Aspartate 199 is the closest negatively charged residue on the receptor to the major determinant of alpha3beta2 selectivity on the toxin, the positively charged histidine 12.

Keywords

Biology, Neuroscience; Health Sciences, Pharmacology; Chemistry, Biochemistry

Link to Full Text

http://access.library.miami.edu/login?url=http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:3168711